Ever since the advent of agriculture thousands of years ago, farmers have been engaged in an ongoing battle to minimize the impact of crop pests. Plant diseases caused by bacteria and fungi are currently a major factor in limiting crop production worldwide. For example, it was estimated that head blight, caused by the fungal pathogens Fusarium graminearum and F. poae caused $3 billion in losses to wheat and barley production in the US between 1991-1996. The impact in less developed countries, where food production is usually at or below sustenance levels, is much more severe. Diseases not only adversely affect overall yield, but have a significant impact on the quality of foods produced. This destruction of crops and decrease in food quality has profound socioeconomic effects, as exemplified by the widespread starvation and subsequent emigration caused by the Irish potato famine of the 1800's. Furthermore, some plant disease agents pose human health hazards, such as mycotoxins produced by fungal phytopathogens.
Fungi are a highly diverse and versatile group of organisms that successfully occupy most natural habitats. Less than 10% of the ˜100,000 known fungal species can colonize plants, and of these, a small fraction are responsible for plant diseases. However, virtually all flowering plants are attacked by and susceptible to some form of phytopathogenic fungus, and the specificity of these interactions is determined by host range limitations of both plant and microbe. In general, fungal phytopathogens may be categorized into three classes: 1) opportunistic parasites, that usually have a broad host range but relatively low virulence, 2) facultative pathogens that rely on living plants to grow but can survive as free-living under some circumstances, and 3) obligate pathogens, for which a living host is an absolute requirement for survival. Many of the most serious and virulent plant disease agents fall into the second class of phytopathogen, the facultative parasites, and it is this group of organisms that is of primary interest to agricultural researchers. Agronomically important diseases caused by fungal phytopathogens include: glume and leaf blotch, late blight, stalk/head rot, rice blast, leaf blight and spot, corn smut, wilt, sheath blight, stem canker, root rot, blackleg and kernel rot.
Plant Defense
Over the course of evolution and natural selection, plants have developed several mechanisms of defense against phytopathogens. This process, often referred to as the “evolutionary arms-race,” continues due to the rapid ability of most pathogens to overcome plant defenses. Plants have several defensive modes-of-action, that often act in concert to mount responses in both generalized and specific manners. Defense mechanisms such as bark, trichomes and waxy cuticles form physical barriers protecting the plant from contact with disease organisms. Additionally, some plants secrete compounds, such as resins or gums, that not only provide a barrier to pathogen contact, but in some cases may act as a repellent.
In addition to physical barriers, plants have developed the ability to mount defense responses when challenged by pathogens. These induced responses require that a plant recognize a pathogen, activate and elaborate a defense pathway, and localize the infection, preventing invasion/spread of the pathogen and full-blown disease. This type of resistant plant-microbe interaction is described as incompatible, since the pathogen is not able to successfully parasitize and infect the host plant. Incompatible interactions involve a complex set of distinct and networked signal transduction pathways, the study of which has been facilitated by molecular analyses of both plant and microbe genes identified in various mutant screens. Generally, the defense pathways induced during incompatible interactions fall into two categories: the hypersensitive response (HR), and systemic acquired resistance (SAR). However, it is clear that there are many intersecting and overlapping branch points in these pathways.
The HR consists of localized, induced cell death in a host plant at the site of pathogen invasion. HR is frequently associated with the appearance of necrotic flecks containing dead plant cells within a few hours of pathogen contact. This plant cell death deprives the pathogen of access to further nutrients, causing pathogen arrest and protecting the rest of the plant from the disease agent. The mechanisms of HR include both the activation of programmed cell death (apoptosis) by the plant and/or a switch in plant cell metabolism, activating biochemical pathways that produce compounds toxic to both pathogen and plant. The triggering of HR is associated with the presence of reactive oxygen species such as superoxide anions and H2O2, that can act as signal molecules in addition to being converted to highly reactive and damaging oxygen radicals. HR is also associated with the induction of benzoic acid (BA), salicylic acid (SA), and their respective glucoside conjugates, which also play signaling roles and may be directly antimicrobial, as well as several classes of PR (pathogen response) proteins.
SAR is a broad-spectrum, inducible plant immunity that is activated after the formation of a necrotic lesion, either as a part of HR or as a symptom of disease. Therefore, it is not limited to incompatible interactions, but may be induced by compatible interactions with disease-causing microbes. This immunity or resistance spreads systemically and develops in distal, unchallenged parts of the plant. SAR acts nonspecifically throughout the plant and reduces the severity of disease symptoms caused by all classes of pathogens, including highly virulent ones. It can be induced by the elicitor SA in a dose-dependent manner, and involves a complex set of signal transduction molecules and downstream elicitors. The SAR response is characterized by the coordinate induction in uninfected leaves of several gene families, including chitinases, β-1,3 glucanases, PR-1 proteins and many others. The exact mechanisms of SAR and HR are still being elucidated, and are also targets for bioengineering of plant disease resistance.
Traditional Agricultural Approaches to Plant Disease Control
Over several centuries of agricultural development, farmers have devised methods for controlling plant disease. Husbandry techniques such as crop rotation, controlled irrigation, manure application, and tilling date back to the Roman Empire. Alone, these methods are limited in their efficacy to control diseases (by modern standards). However, they are still considered standard practice, and contribute significantly to any comprehensive pest management program.
In addition to husbandry, breeding methods have been employed to develop disease-resistant cultivars. The ability to select and propagate cultivars of crops containing desirable traits has enabled plant breeders to take advantage of natural genetic variation and/or induced mutations. There are numerous genetic methods and techniques available to breeders, including crossing and hybridization, embryo rescue, cell fusion and mutagenesis. The programs breeders implement depend on both the type of cultivar they want to improve (e.g., hybrid vs. inbred) and the reproductive biology of the particular species (self-pollinated vs. out-crossed). One example of a successful breeding program is that of blight-resistant potatoes that are a result of introducing traits from a Mexican species into >50% of all cultivars. Conventional breeding methods will undoubtedly continue to play a significant role in the improvement of agricultural crops, however, the time-scale and labor requirements of breeding programs may not be adequate to meet increasing demands for many agronomic traits, including disease resistance. Furthermore, the ability of pathogens to rapidly overcome resistance bred into new races of plants limits the utility and useful lifetime of these crops.
Within the last several decades, agricultural techniques have expanded to include widespread and intensive use of chemicals. A recent study of US farm-sector sales of pesticides estimated that for 11 major crops, a total of approximately $8.83 billion was spent in 1997 alone. This represents a significant portion of the US agriculture economy. In addition to chemical control, bio-control methods have gained a smaller, but constantly growing, following among farmers. As concern for the global environment and human health increases, it is imperative that new agricultural practices be developed and implemented.
AgBiotech Approaches to Plant Disease Control
The advent of modern biology, particularly molecular biology and genetics, has opened up new avenues for disease control research and practice. Scientists have focused on utilizing recombinant DNA (rDNA) methods, which allow new varieties of plants to be produced much faster than by conventional breeding. rDNA techniques allow the introduction of genes from distantly related species or even from different biological kingdoms into crop plants, conferring traits that provide significant agronomic advantages. Furthermore, detailed knowledge of the traits being introduced, such as cellular function and localization, can lead to less variability in offspring, and fine-tuning of secondary effects. After a trait has been introduced into a plant by transgenic methods, conventional breeding can be used to hybridize the transgenic line with useful varieties and elite germplasms, resulting in crops containing numerous advantageous properties.
Agricultural biotechnology (AgBiotech) approaches to disease resistance are typically three-fold. First, specific crops that undergo compatible (disease causing) interactions with specific pathogens are analyzed to determine the endogenous factors that enable this interaction, in an effort to prevent the particular disease(s) via bio-engineering. Second, researchers look for exogenous factors (compounds and proteins) from other species/sources that, when produced in crop plants, provide protection from phytopathogens. Finally, efforts are being made to hyper-activate the plant's own defense responses, in order to provide crops with broad-spectrum immunity against several disease agents simultaneously. Each of these approaches has it's advantages and disadvantages, and has met with some limited success to date. However, intensive research and testing continues; between 1987 and May 1999, there were 61 publicly-sponsored and 272 privately-sponsored field trials testing genes for fungal disease resistance in transgenic crops. A recent example of a successful disease-resistance bioengineered product was described by the Monsanto Company, which demonstrated that a potato engineered to express an alfalfa antifungal peptide (defensin) showed robust resistance to the fungal pathogen Verticillium dahliae (Verticillium wilt) under both greenhouse and field conditions.
As AgBiotech hurtles into the genomics and post-genomics era, the massive amounts of genetic and functional data being generated are being used to direct the search for genes that can be utilized with recombinant methods. Additionally, transgenic technology itself is overcoming some of it's rate-limiting obstacles, allowing expression and modulation of several genes simultaneously in transgenic crops. These advances in both the informational and technological tools available to agricultural biotechnologists has and will continue to increase the pace of discovery and product development with regards to disease resistance. As the regulatory and commercial framework is developed, many of these AgBiotech products will be entering the marketplace. It is therefore reasonable to expect that in the very near future, bioengineered crops will be part of a comprehensive, integrated disease management program throughout the agricultural enterprise.
Accordingly, what is needed in the art are gene sequences and polypeptide sequences whose expression in plants, plant seeds, plant tissues and/or plant cells causes resistance to plant pathogens.